Muscarinic acetylcholine receptors (mAChRs) are a family of at least four structurally distinct subtypes which regulate a variety of physiological and biochemical responses throughout the central and autonomic nervous systems. The proposed research will employ techniques of molecular genetics to study the interactions between recombinant mAChR subtypes, the adenylyl cyclase and phosphoinositide biochemical effector pathways, and ion channels in tissue culture expression systems. These studies will provide important information regarding the mechanisms by which individual mAChR subtypes interact with G proteins to regulate central nervous activities and control the rate and force of heart muscle contraction. The structural domains involved in determining the specificity of G protein activation by mAChR subtypes are unknown. To determine the regions of these receptors involved in G protein recognition and activation, a series of hybrid receptors composed of domains derived from functionally distinct Ml and M2 mAChR subtypes will be constructed and expressed within stably transfected mammalian cell lines. The Ml subtype potently stimulates PI hydrolysis and fails to inhibit adenylyl cyclase, while the M2 subtype efficiently inhibits adenylyl cyclase and weakly increases PI hydrolysis. Each hybrid receptor will be tested for its ability to bind muscarinic ligands, inhibit adenylyl cyclase, stimulate PI hydrolysis, and release intracellular calcium. The effects of pertussis toxin on these responses will also be investigated. These studies will identify important domains through gain, rather than loss, of function. The mechanisms of ion channel regulation by individual mAChR subtypes are poorly understood. To determine the ability of individual subtypes to regulate ion channel activity, molecular clones encoding acetylcholine-sensitive potassium channels will be isolated and expressed in the presence of defined mAChR subtypes and G proteins. In particular, molecular clones encoding the atrial inward rectifying potassium channel will be isolated to study the novel regulation of this channel by mAChRs. The effect of muscarinic stimulation on channel activity will be analyzed by patch clamp of transfected mammalian cells and voltage clamp of Xenopus oocytes injected with mRNAs encoding potassium channel and mAChR proteins. Ultimately, mutagenesis studies will be conducted to determine the important structures involved in the regulation of potassium channels by mAChRs and G proteins.